The burden of Plasmodium falciparum malaria is enormous with up to a million deaths per annum and billions of infections that persist for many months in the human population. These chronic infections, largely asymptomatic, constitute the reservoir of infection and serve to fuel continued malaria transmission. Traditionally, the reservoir of infection has been measured in population surveys by microscopic visualization of the parasite in blood smears. This method, however, is not sensitive and does not capture within species variation to accurately assess the true reservoir of P. falciparum where individuals in malaria endemic areas carry multiple distinct parasite genomes. The var multigene family encodes P. falciparum Erythrocyte Membrane Protein-1 (PfEMP1), a molecule that is expressed on the surface of erythrocytes infected by asexual parasite stages and transmission stages. There are 50-60 var genes in a P. falciparum genome and differential expression of these genes underlies clonal antigenic variation. This ability to switch surface antigen variants allows the malaria parasite to evade the host immune response and establish chronic infection to optimize transmission to the mosquito. Diverse repertoires of var genes exist in different parasite genomes. This repertoire diversity is proposed to allow a superinfection to establish, persist, and transmit in an exposed host. Thus, var gene diversity gives a specific diagnostic measure of the size as well as the potential for persistence or duration of the reservoir of infection in an endemic area. The overall goal of this proposal is to understand the evolution of var genes in order to improve molecular surveillance of P. falciparum. To achieve this goal, we need to collect baseline var diversity data in three global settings: sub- Saharan Africa, Melanesia, and South America. Each of these settings displays a distinct epidemiology of malaria due to differences including vector biology, transmission intensity and population history. We will use a standardized molecular epidemiology framework with custom made statistics and bioinformatics developed over the past 6 years by the Day laboratory to describe the population genetics of var genes. In addition, we will analyze microsatellite diversity as a marker for local parasite population structure. The important hypothesis--that geographic variation in var diversity exists at a local level relative to global level--will be tested as such population structure would influence cross-immunity among parasite populations. Moreover, such structure would dramatically influence the approach to developing and assessing elimination and eradication strategies to reduce the reservoir of malaria infection in a more sophisticated manner than existing methods. This is an important interdisciplinary natural history experiment, akin to defining variation in the hemagglutinin molecule for influenza disease surveillance, except that the var genetics is more complex due to the multiple, highly diverse var copies per genome and possibilities for recombination. Defining the reservoir of infection by measuring var diversity is a critical step for malaria control in the new era of malaria eradication.